The molecular mechanisms underlying uveitis (intraocular inflammation) are poorly understood, a major barrier to progress in treating the disease or designing specific therapeutics. This proposal builds on our exciting discovery that Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV; a type of uveitis) can be caused by mutations in the cysteine protease CAPN5 (calpain-5), one member of a family of proteins that uses proteolysis to control a spectrum of molecular events. Thus, CAPN5 is the first nonsyndromic uveitis gene to be identified, which now makes possible a highly innovative molecular-genetic approach to identifying the factors responsible for intraocular inflammation, neovascularization, and fibrosis. The long-term goals of our research are to identify new therapeutic targets for uveitis. Our objective in this proposal is to identify the structure-functin mechanism of calpain-5 mutations that activate molecular pathways leading to uveitis and probe inhibitor interactions. Our central hypothesis is that ADNIV mutations increase calpain-5 proteolytic activity and are amenable to structure guided inhibition that will eventually be used t prevent retinal inflammation. Our rationale is that, intracellular calpain-5 activity is normally a tightly controlled proteolytic switch for activating signaling molecules, and when calpain-5 is hyperactive, target signals are tripped pathogenically. Excess calpain activity has been implicated in the pathogenesis of a wide range of human diseases including cancer, multiple sclerosis, Alzheimer's disease, diabetes, and muscular dystrophy. Although little is known about CAPN5, our preliminary studies suggest ADNIV disease mutants increase CAPN5 enzymatic activity through structural changes and alter cellular transcription. Our specific aims are to test the hypotheses that: (1) ADNIV mutants alter CAPN5 catalytic and proteolytic activity in vitro; (2) CAPN5 and ADNIV residues affect activity of other calpains; and (3) ADNIV mutations alter CAPN5 structure using circular dichroism spectroscopy, small angle x-ray scattering SAXS, and crystallization. We will use novel enzymatic methods and modeling to pursue studies that trace the molecular mechanism of CAPN5 mutations. Our work should have a significant positive impact on identifying new molecular mechanisms for therapeutic intervention of uveitis. At the completion of these experiments, we expect to have in hand a CAPN5 catalytic activity assays that can ultimately be used for low and high-throughput drug screening. We also expect to resolve the structure of the protein, which will expose differences between CAPN5 and its orthologs. Since the ADNIV phenocopies other eye diseases, our studies promise to have a broad positive impact, beyond ADNIV and uveitis patients, where components of the CAPN5 pathway may be therapeutic targets.